Ae4 (Slc4a9) Anion Exchanger Drives Cl? Uptake-dependent Fluid Secretion by Mouse Submandibular Gland Acinar Cells

Transcellular Cl⁻ movement across acinar cells is the rate-limiting step for salivary gland fluid secretion. Basolateral Nkcc1 Na⁺-K⁺-2Cl⁻ cotransporters play a critical role in fluid secretion by promoting the intracellular accumulation of Cl⁻ above its equilibrium potential. However, salivation is only partially abolished in the absence of Nkcc1 cotransporter activity, suggesting that another Cl⁻ uptake pathway concentrates Cl⁻ ions in acinar cells. To identify alternative molecular mechanisms, we studied mice lacking Ae2 and Ae4 Cl⁻/HCO₃⁻ exchangers. We found that salivation stimulated by muscarinic and β-adrenergic receptor agonists was normal in the submandibular glands of Ae2^−/− mice. In contrast, saliva secretion was reduced by 35% in Ae4^−/− mice. The decrease in salivation was not related to loss of Na⁺-K⁺-2Cl⁻ cotransporter or Na⁺/H⁺ exchanger activity in Ae4^−/− mice but correlated with reduced Cl⁻ uptake during β-adrenergic receptor activation of cAMP signaling. Direct measurements of Cl⁻/HCO₃⁻ exchanger activity revealed that HCO₃⁻-dependent Cl⁻ uptake was reduced in the acinar cells of Ae2^−/− and Ae4^−/− mice. Moreover, Cl⁻/HCO₃⁻ exchanger activity was nearly abolished in double Ae4/Ae2 knock-out mice, suggesting that most of the Cl⁻/HCO₃⁻ exchanger activity in submandibular acinar cells depends on Ae2 and Ae4 expression. In conclusion, both Ae2 and Ae4 anion exchangers are functionally expressed in submandibular acinar cells; however, only Ae4 expression appears to be important for cAMP-dependent regulation of fluid secretion.     

Intracellular pH Regulation in Cultured Astrocytes from Rat Hippocampus : II. Electrogenic Na/HCO3 Cotransport

In the preceding paper (Bevensee, M.O., R.A. Weed, and W.F. Boron. 1997. J. Gen. Physiol. 110: 453–465.), we showed that a Na⁺-driven influx of HCO₃ ⁻ causes the increase in intracellular pH (pH_i) observed when astrocytes cultured from rat hippocampus are exposed to 5% CO₂/17 mM HCO₃ ⁻. In the present study, we used the pH-sensitive fluorescent indicator 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and the perforated patch-clamp technique to determine whether this transporter is a Na⁺-driven Cl-HCO₃ exchanger, an electrogenic Na/HCO₃ cotransporter, or an electroneutral Na/HCO₃ cotransporter. To determine if the transporter is a Na⁺-driven Cl-HCO₃ exchanger, we depleted the cells of intracellular Cl⁻ by incubating them in a Cl⁻-free solution for an average of ∼11 min. We verified the depletion with the Cl⁻-sensitive dye N-(6-methoxyquinolyl)acetoethyl ester (MQAE). In Cl⁻-depleted cells, the pH_i still increases after one or more exposures to CO₂/HCO₃ ⁻. Furthermore, the pH_i decrease elicited by external Na⁺ removal does not require external Cl⁻. Therefore, the transporter cannot be a Na⁺-driven Cl-HCO₃ exchanger. To determine if the transporter is an electrogenic Na/ HCO₃ cotransporter, we measured pH_i and plasma membrane voltage (V_m) while removing external Na⁺, in the presence/absence of CO₂/HCO₃ ⁻ and in the presence/absence of 400 μM 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS). The CO₂/HCO₃ ⁻ solutions contained 20% CO₂ and 68 mM HCO₃ ⁻, pH 7.3, to maximize the HCO₃ ⁻ flux. In pH_i experiments, removing external Na⁺ in the presence of CO₂/HCO₃ ⁻ elicited an equivalent HCO₃ ⁻ efflux of 281 μM s⁻¹. The HCO₃ ⁻ influx elicited by returning external Na⁺ was inhibited 63% by DIDS, so that the predicted DIDS-sensitive V_m change was 3.3 mV. Indeed, we found that removing external Na⁺ elicited a DIDS-sensitive depolarization that was 2.6 mV larger in the presence than in the absence of CO₂/ HCO₃ ⁻. Thus, the Na/HCO₃ cotransporter is electrogenic. Because a cotransporter with a Na⁺:HCO₃ ⁻ stoichiometry of 1:3 or higher would predict a net HCO₃ ⁻ efflux, rather than the required influx, we conclude that rat hippocampal astrocytes have an electrogenic Na/HCO₃ cotransporter with a stoichiometry of 1:2.     

Intracellular Cl? Dependence of Na-H Exchange in Barnacle Muscle Fibers under Normotonic and Hypertonic Conditions

We previously showed that shrinking a barnacle muscle fiber (BMF) in a hypertonic solution (1,600 mosM/kg) stimulates an amiloride-sensitive Na-H exchanger. This activation is mediated by a G protein and requires intracellular Cl⁻. The purpose of the present study was to determine (a) whether Cl⁻ plays a role in the activation of Na-H exchange under normotonic conditions (975 mosM/kg), (b) the dose dependence of [Cl⁻]_i for activation of the exchanger under both normo- and hypertonic conditions, and (c) the relative order of the Cl⁻- and G-protein-dependent steps. We acid loaded BMFs by internally dialyzing them with a pH-6.5 dialysis fluid containing no Na⁺ and 0–194 mM Cl⁻. The artificial seawater bathing the BMF initially contained no Na⁺. After dialysis was halted, adding 50 mM Na⁺ to the artificial seawater caused an amiloride-sensitive pH_i increase under both normo- and hypertonic conditions. The computed Na-H exchange flux (J _Na-H) increased with increasing [Cl⁻]_i under both normo- and hypertonic conditions, with similar apparent K _m values (∼120 mM). However, the maximal J _Na-H increased by nearly 90% under hypertonic conditions. Thus, activation of Na-H exchange at low pH_i requires Cl⁻ under both normo- and hypertonic conditions, but at any given [Cl⁻]_i, J _Na-H is greater under hyper- than normotonic conditions. We conclude that an increase in [Cl⁻]_i is not the primary shrinkage signal, but may act as an auxiliary shrinkage signal. To determine whether the Cl⁻-dependent step is after the G-protein-dependent step, we predialyzed BMFs to a Cl⁻-free state, and then attempted to stimulate Na-H exchange by activating a G protein. We found that, even in the absence of Cl⁻, dialyzing with GTPγS or AlF₃, or injecting cholera toxin, stimulates Na-H exchange. Because Na-H exchange activity was absent in control Cl⁻-depleted fibers, the Cl⁻-dependent step is at or before the G protein in the shrinkage signal-transduction pathway. The stimulation by AlF₃ indicates that the G protein is a heterotrimeric G protein.     

Vasopressin alters the mechanism of apical Cl− entry from Na+:Cl− to Na+:K+:2Cl− cotransport in mouse medullary thick ascending limb

Summary Experiments were performed usingin vitro perfused medullary thick ascending limbs of Henle (MTAL) and in suspensions of MTAL tubules isolated from mouse kidney to evaluate the effects of arginine vasopressin (AVP) on the K⁺ dependence of the apical, furosemide-sensitive Na⁺:Cl^– cotransporter and on transport-related oxygen consumption (QO₂). In isolated perfused MTAL segments, the rate of cell swelling induced by removing K⁺ from, and adding onemm ouabain to, the basolateral solution [ouabain(zero-K⁺)] provided an index to apical cotransporter activity and was used to evaluated the ionic requirements of the apical cotransporter in the presence and absence of AVP. In the absence of AVP cotransporter activity required Na⁺ and Cl^–, but not K⁺, while in the presence of AVP the apical cotransporter required all three ions.⁸⁶Rb⁺ uptake into MTAL tubules in suspension was significant only after exposure of tubules to AVP. Moreover,²²Na⁺ uptake was unaffected by extracellular K⁺ in the absence of AVP while after AVP exposure²²Na⁺ uptake was strictly K⁺-dependent. The AVP-induced coupling of K⁺ to the Na⁺:Cl^– cotransporter resulted in a doubling in the rate of NaCl absorption without a parallel increase in the rate of cellular²²Na⁺ uptake or transport-related oxygen consumption. These results indicate that arginine vasopressin alters the mode of a loop diuretic-sensitive transporter from Na⁺:Cl^– cotransport to Na⁺:K⁺:2Cl^– cotransport in the mouse MTAL with the latter providing a distinct metabolic advantage for sodium transport. A model for AVP action on NaCl absorption by the MTAL is presented and the physiological significance of the coupling of K⁺ to the apical Na⁺:Cl^– cotransporter in the MTAL and of the enhanced metabolic efficiency are discussed.     

Solute Accumulation in Tobacco Cells Adapted to NaCl

Cells of Nicotiana tabacum L. var Wisconsin 38 adapted to NaCl (up to 428 millimolar) which have undergone extensive osmotic adjustment accumulated Na⁺ and Cl⁻ as principal solutes for this adjustment. Although the intracellular concentrations of Na⁺ and Cl⁻ correlated well with the level of adaptation, these ions apparently did not contribute to the osmotic adjustment which occurred during a culture growth cycle, because the concentrations of Na⁺ and Cl⁻ did not increase during the period of most active osmotic adjustment. The average intracellular concentrations of soluble sugars and total free amino acids increased as a function of the level of adaptation; however, the levels of these solutes did not approach those observed for Na⁺ and Cl⁻. The concentration of proline was positively correlated with cell osmotic potential, accumulating to an average concentration of 129 millimolar in cells adapted to 428 millimolar NaCl and representing about 80% of the total free amino acid pool as compared to an average of 0.29 millimolar and about 4% of the pool in unadapted cells. These results indicate that although Na⁺ and Cl⁻ are principal components of osmotic adjustment, organic solutes also may make significant contributions.     

Maintenance of low cl concentrations in mesophyll cells of leaf blades of barley seedlings exposed to salt stress

The concentrations of vacuolar Na⁺ and Cl⁻ in the epidermal and mesophyll cells of the leaf blade and sheath of Hordeum vulgare seedlings (cv California Mariout and Clipper) were measured by means of quantitative electron probe x-ray microanalysis. A preferential accumulation of Cl⁻ in vacuoles of epidermal cells in both blade and sheath and a low level in mesophyll cells of the blade were evident in plants grown in full strength Johnson solution. The concentration of Cl⁻ in the mesophyll cells of the blade remained at a low level after exposure to 50 or 100 millimolar NaCl for 1 day or to 50 millimolar for 4 days, while at the same time the concentration of Cl⁻ in the epidermis and mesophyll of the sheath showed a dramatic increase. Clipper generally contained more Cl⁻ in the mesophyll cells of the blade than California Mariout. A greater accumulation of Na⁺ in the mesophyll of the sheath relative to that of the blade was only apparent after treatment with 100 millimolar NaCl for 1 day or 50 millimolar for 4 days. These results confirm the suggestion that sheath tissue is capable of accumulating excess Cl⁻ (and to a lesser extent Na⁺) and suggest that the site of regulation of Cl⁻ concentration in the barley leaf is located in the mesophyll cells of the blade.     

Relation between Ion Accumulation of Salt-Sensitive and Isolated Stable Salt-Tolerant Cell Lines of Citrus aurantium

Four selected NaCl-tolerant cell lines of Sour orange (Citrus aurantium) were compared with the nonselected cell line in their growth and internal ion content of Na⁺, K⁺, and Cl⁻ when exposed to increasing NaCl concentrations. No difference was found among the various NaCl-tolerant cell lines in Na⁺ and Cl⁻ uptake, and all these cell lines took up similar or even larger amounts of Na⁺ and Cl⁻ than the NaCl-sensitive cell line. Exposure of cells of NaCl-sensitive and NaCl-tolerant lines to equal external concentrations of NaCl, resulted in a greater loss of K⁺ from the NaCl-sensitive cell line. This observation leads to the conclusion that growth and ability to retain high levels of internal K⁺ are correlated. Exposure of the NaCl-tolerant cell lines to salts other than NaCl resulted in even greater tolerance to Na₂SO₄, but rather poor tolerance to K⁺ introduced as either K₂SO₄ or KCl; the latter has a stronger inhibitory effect. The NaCl-sensitive cell line proved to be more sensitive to replacement of Na⁺ by K⁺. Analyses of internal Na⁺, K⁺, and Cl⁻ concentrations failed to identify any particular internal ion concentration which could serve as a reliable marker for salt tolerance.     

Inhibition of cAMP-Activated Intestinal Chloride Secretion by Diclofenac: Cellular Mechanism and Potential Application in Cholera

Cyclic AMP-activated intestinal Cl⁻ secretion plays an important role in pathogenesis of cholera. This study aimed to investigate the effect of diclofenac on cAMP-activated Cl⁻ secretion, its underlying mechanisms, and possible application in the treatment of cholera. Diclofenac inhibited cAMP-activated Cl⁻ secretion in human intestinal epithelial (T84) cells with IC₅₀ of ∼20 µM. The effect required no cytochrome P450 enzyme-mediated metabolic activation. Interestingly, exposures of T84 cell monolayers to diclofenac, either in apical or basolateral solutions, produced similar degree of inhibitions. Analyses of the apical Cl⁻ current showed that diclofenac reversibly inhibited CFTR Cl⁻ channel activity (IC₅₀∼10 µM) via mechanisms not involving either changes in intracellular cAMP levels or CFTR channel inactivation by AMP-activated protein kinase and protein phosphatase. Of interest, diclofenac had no effect on Na⁺-K⁺ ATPases and Na⁺-K⁺-Cl⁻ cotransporters, but inhibited cAMP-activated basolateral K⁺ channels with IC₅₀ of ∼3 µM. In addition, diclofenac suppressed Ca²⁺-activated Cl⁻ channels, inwardly rectifying Cl⁻ channels, and Ca²⁺-activated basolateral K⁺ channels. Furthermore, diclofenac (up to 200 µM; 24 h of treatment) had no effect on cell viability and barrier function in T84 cells. Importantly, cholera toxin (CT)-induced Cl⁻ secretion across T84 cell monolayers was effectively suppressed by diclofenac. Intraperitoneal administration of diclofenac (30 mg/kg) reduced both CT and Vibrio cholerae-induced intestinal fluid secretion by ∼70% without affecting intestinal fluid absorption in mice. Collectively, our results indicate that diclofenac inhibits both cAMP-activated and Ca²⁺-activated Cl⁻ secretion by inhibiting both apical Cl⁻ channels and basolateral K⁺ channels in intestinal epithelial cells. Diclofenac may be useful in the treatment of cholera and other types of secretory diarrheas resulting from intestinal hypersecretion of Cl⁻.     

Effect of Salinity upon Cell Membrane Potential in the Marine Halophyte, Salicornia bigelovii Torr

The electrophysiology of root cells of the marine halophyte, Salicornia bigelovii Torr., has been investigated. Cellular concentrations of K⁺, Cl⁻, and Na⁺ and resulting cell membrane potentials were determined as functions of time and exposure to dilutions of artificial seawater. Treatment of these data by the Nernst criterion suggests that Cl⁻ is actively transported into these root cells, but that active transport need not be invoked to explain the accumulation of Na⁺ at all salinities investigated nor for K⁺ at moderate to high salinities. In low environmental salinity, the cell electropotential of Salicornia root cells was found to respond to inhibitors in a fashion similar to that observed in glycophytes; in high environmental salinity, root cell membrane potential appears to be insensitive to bathing salinity and m-chlorocarbonylcyanide phenylhydrazone induces membrane hyperpolarization, in contrast to the response of glycophytes to such treatments. The fact that measured membrane potentials exceed diffusion potentials for Na⁺, K⁺, and Cl⁻ and the observation of a rapid depolarization by CO in the dark suggests an electrogenic component in Salicornia root cell membrane potentials.     

Inability of Ehrlich ascites tumor cells to volume regulate following a hyperosmotic challenge

Summary Ehrlich cells shrink when the osmolality of the suspending medium is increased and behave, at least initially, as osmometers. Subsequent behavior depends on the nature of the hyperosmotic solute but in no case did the cells exhibit regulatory volume increase. With hyperosmotic NaCl an osmometric response was found and the resultant volume maintained relatively constant. Continuous shrinkage was observed, however, with sucrose-induced hyperosmolality. In both cases increasing osmolality from 300 to 500 mOsm initiated significant changes in cellular electrolyte content, as well as intracellular pH. This was brought about by activation of the Na⁺/H⁺ exchanger, the Na/K pump, the Na⁺+K⁺+2Cl^– cotransporter and by loss of K⁺ via a Ba-sensitive pathway. The cotransporter in response to elevated [Cl^–] _i (100mm) and/or the increase in the outwardly directed gradient of chemical potential for Na⁺, K⁺ and Cl^–, mediated net loss of ions which accounted for cell shrinkage in the sucrose-containing medium. In hyperosmotic NaCl, however, the net Cl^– flux was almost zero suggesting minimal net cotransport activity.We conclude that volume stability following cell shrinkage depends on the transmembrane gradient of chemical potential for [Na⁺+K⁺+Cl^–], as well as the ratio of intra- to extracellular [Cl^–]. Both factors appear to influence the activity of the cotransport pathway.     

Physiological Control of Chloride Transport in Chara corallina: II. THE ROLE OF CHLORIDE AS A VACUOLAR OSMOTICUM

The extent to which Cl⁻ is replaceable as the major anionic constituent of the vacuole of Chara corallina was investigated. It was found that external Cl⁻ is not essential in order for nongrowing cells to increase internal osmotic pressure. After growth of cells in low (9 micromolar) Cl⁻, the vacuolar Cl⁻ concentration is one-half that of cells grown at normal external Cl⁻ concentration (850 micromolar). In contrast, both internal osmotic pressure and total concentration of the major cations, K⁺ and Na⁺, in the same cells were found to be only slightly sensitive to the external Cl⁻ concentration. Thus, it is proposed that, at limiting external Cl⁻ concentration, the cell is able to transport or synthesize another anion for vacuolar use rather than utilize a neutral solute.     

Functional expression of the murine D2, D3 and D4 dopamine receptors in Xenopus laevis oocytes

The different murine D2-type dopamine receptors (D_2L, D_2S, D_3L, D_3S, and D₄) were expressed in Xenopus laevis oocytes. The D2-type receptors were all similarly and efficiently expressed in Xenopus oocytes and were shown to bind the D2 antagonist [¹²⁵I]sulpride. They were all shown to activate Cl⁻ influx upon agonist stimulation. Using the diagnostic inhibitor bumetanide, we were able to separate the Na⁺/K⁺/2Cl⁻ cotransporter component of the Cl⁻ influx from the total unidirectional Cl⁻ influx. The D_3L subtype was found to operate exclusively through the bumetanide-insensitive Cl⁻ influx whereas the other D2-type receptors acted on the Na⁺/K⁺/2Cl⁻ cotransporter as well. The pertussis toxin sensitivity of the receptor-activated chloride influx via the Na⁺/K⁺/2Cl⁻ cotransporter varied between the various D2-type receptors showing that they may couple to different G proteins, and activate different second messenger systems.     

Trigeminal Ganglion Neurons of Mice Show Intracellular Chloride Accumulation and Chloride-Dependent Amplification of Capsaicin-Induced Responses

Intracellular Cl⁻ concentrations ([Cl⁻]_i) of sensory neurons regulate signal transmission and signal amplification. In dorsal root ganglion (DRG) and olfactory sensory neurons (OSNs), Cl⁻ is accumulated by the Na⁺-K⁺-2Cl⁻ cotransporter 1 (NKCC1), resulting in a [Cl⁻]_i above electrochemical equilibrium and a depolarizing Cl⁻ efflux upon Cl⁻ channel opening. Here, we investigate the [Cl⁻]_i and function of Cl⁻ in primary sensory neurons of trigeminal ganglia (TG) of wild type (WT) and NKCC1^−/− mice using pharmacological and imaging approaches, patch-clamping, as well as behavioral testing. The [Cl⁻]_i of WT TG neurons indicated active NKCC1-dependent Cl⁻ accumulation. Gamma-aminobutyric acid (GABA)_A receptor activation induced a reduction of [Cl⁻]_i as well as Ca²⁺ transients in a corresponding fraction of TG neurons. Ca²⁺ transients were sensitive to inhibition of NKCC1 and voltage-gated Ca²⁺ channels (VGCCs). Ca²⁺ responses induced by capsaicin, a prototypical stimulus of transient receptor potential vanilloid subfamily member-1 (TRPV1) were diminished in NKCC1^−/− TG neurons, but elevated under conditions of a lowered [Cl⁻]_o suggesting a Cl⁻-dependent amplification of capsaicin-induced responses. Using next generation sequencing (NGS), we found expression of different Ca²⁺-activated Cl⁻ channels (CaCCs) in TGs of mice. Pharmacological inhibition of CaCCs reduced the amplitude of capsaicin-induced responses of TG neurons in Ca²⁺ imaging and electrophysiological recordings. In a behavioral paradigm, NKCC1^−/− mice showed less avoidance of the aversive stimulus capsaicin. In summary, our results strongly argue for a Ca²⁺-activated Cl⁻-dependent signal amplification mechanism in TG neurons that requires intracellular Cl⁻ accumulation by NKCC1 and the activation of CaCCs.     

Compartments and Fluxes of K, NA, and CL in Avena Coleoptile Cells

By the compartmental analysis method of MacRobbie and Dainty, and Pitman, estimates of K⁺, Na⁺, and Cl⁻ concentrations and fluxes were obtained for the cytoplasm and vacuole of coleoptile cells of oat, Avena sativa L. cv. Victory. Double labeling was used in experiments with ⁴²K plus ²²Na and with ⁴²K plus ³⁶Cl in a complete nutrient solution. At the plasmalemma, according to the Ussing-Teorell flux ratio equation, Na⁺ is pumped out and Cl⁻ is actively transported inward. The results with K⁺ are less conclusive, but it is probably pumped in. At the tonoplast there is an active inward transport of Na⁺ and probably of K⁺, but the status of Cl⁻ is uncertain, depending upon whether there is an electrical potential difference between the cytoplasm and vacuole. The results suggest that ion selectivity resides mostly in the plasmalemma. Possible errors in the estimates and interpretations are discussed.     

Characterization of a Cyanobacterial Chloride-pumping Rhodopsin and Its Conversion into a Proton Pump

Light-driven ion-pumping rhodopsins are widely distributed in microorganisms and are now classified into the categories of outward H⁺ and Na⁺ pumps and an inward Cl⁻ pump. These different types share a common protein architecture and utilize the photoisomerization of the same chromophore, retinal, to evoke photoreactions. Despite these similarities, successful pump-to-pump conversion had been confined to only the H⁺ pump bacteriorhodopsin, which was converted to a Cl⁻ pump in 1995 by a single amino acid replacement. In this study we report the first success of the reverse conversion from a Cl⁻ pump to a H⁺ pump. A novel microbial rhodopsin (MrHR) from the cyanobacterium Mastigocladopsis repens functions as a Cl⁻ pump and belongs to a cluster that is far distant from the known Cl⁻ pumps. With a single amino acid replacement, MrHR is converted to a H⁺ pump in which dissociable residues function almost completely in the H⁺ relay reactions. MrHR most likely evolved from a H⁺ pump, but it has not yet been highly optimized into a mature Cl⁻ pump.     

Diffusion and Electric Mobility of Ions within Isolated Cuticles of Citrus aurantium: Steady-State and Equilibrium Values

We report a new method for measuring cation and anion permeability across cuticles of sour orange, Citrus aurantium, leaves. The method requires the measurement of two electrical parameters: the diffusion potential arising when the two sides of the cuticle are bathed in unequal concentrations of a Cl⁻ salt; and the electrical conductance of the cuticle measured at a salt concentration equal to the average of that used in the diffusion-potential measurement. The permeabilities of H⁺, Li⁺, Na⁺, K⁺, and Cs⁺ ranged from 2 × 10⁻⁸ to 0.6 × 10⁻⁸ meters per second when cuticles were bathed in 2 moles per cubic meter Cl⁻ salts. The permeability of Cl⁻ was 3 × 10⁻⁹ meters per second. The permeability of Li⁺, Na⁺, and K⁺ was about five times less when measured in 500 moles per cubic meter Cl⁻ salts. We also report an asymmetry in cuticle-conductance values depending on the magnitude and the direction of current flow. The asymmetry disappears at low current-pulse magnitude and increases linearly with the magnitude of the current pulse. This phenomenon is explained in terms of transport-number effects in a bilayer model of the cuticle. Conductance is not augmented by current carried by exchangeable cations in cuticles; conductance is rate limited by the outer waxy layer of the cuticle.     

Na+, K+, Cl− cotransport and its regulation in Ehrlich ascites Tumor cells. Ca2+/Calmodulin and protein kinase C dependent pathways

Summary Net Cl^– uptake as well as unidirectional³⁶Cl influx during regulatory volume increase (RVI) require external K⁺. Half-maximal rate of bumetanide-sensitive³⁶Cl uptake is attained at about 3.3mm external K⁺. The bumetanide-sensitive K⁺ influx found during RVI is strongly dependent on both Na⁺ and Cl^–. The bumetanide-sensitive unidirectional Na⁺ influx during RVI is dependent on K⁺ as well as on Cl^–. The cotransporter activated during RVI in Ehrlich cells, therefore, seems to transport Na⁺, K⁺ and Cl^–. In the presence of ouabain and Ba⁺ the stoichiometry of the bumetanide-sensitive net fluxes can be measured at 1.0 Na⁺, 0.8 K⁺, 2.0 Cl^– or approximately 1 : Na, 1 : K, 2 : Cl. Under these circumstances the K⁺ and Cl^– flux ratios (influx/efflux) for the bumetanide-sensitive component were estimated at 1.34 ±0.08 and 1.82 ± 0.15 which should be compared to the gradient for the Na⁺, K⁺, 2Cl^– cotransport system at 1.75 ± 0.24.Addition of sucrose to hypertonicity causes the Ehrlich cells to shrink with no signs of RVI, whereas shrinkage with hypertonic standard medium (all extracellular ion concentrations increased) results in a RVI response towards the original cell volume. Under both conditions a bumetanide-sensitive unidirectional K⁺ influx is activated. During hypotonic conditions a small bumetanide-sensitive K⁺ influx is observed, indicating that the cotransport system is already activated.The cotransport is activated 10–15 fold by bradykinin, an agonist which stimulates phospholipase C resulting in release of internal Ca²⁺ and activation of protein kinase C.The anti-calmodulin drug pimozide inhibits most of the bumetanide-sensitive K⁺ influx during RVI. The cotransporter can be activated by the phorbol ester TPA. These results indicate that the stimulation of the Na⁺, K⁺, Cl^– cotransport involves both Ca²⁺/calmodulin and protein kinase C.     

Intracellular compartmentation of ions in salt adapted tobacco cells

Na⁺ and Cl⁻ are the principal solutes utilized for osmotic adjustment in cells of Nicotiana tabacum L. var Wisconsin 38 (tobacco) adapted to NaCl, accumulating to levels of 472 and 386 millimolar, respectively, in cells adapted to 428 millimolar NaCl. X-ray microanalysis of unetched frozen-hydrated cells adapted to salt indicated that Na⁺ and Cl⁻ were compartmentalized in the vacuole, at concentrations of 780 and 624 millimolar, respectively, while cytoplasmic concentrations of the ions were maintained at 96 millimolar. The morphometric differences which existed between unadapted and salt adapted cells, (cytoplasmic volume of 22 and 45% of the cell, respectively), facilitated containment of the excited volume of the x-ray signal in the cytoplasm of the adapted cells. Confirmation of ion compartmentation in salt adapted cells was obtained based on kinetic analyses of ²²Na⁺ and ³⁶Cl⁻ efflux from cells in steady state. These data provide evidence that ion compartmentation is a component of salt adaptation of glycophyte cells.     

Effects of Helminthosporium carbonum Toxin on Absorption of Solutes by Corn Roots

Susceptible corn roots exposed to the host-selective toxin of Helminthosporium carbonum took up and retained more NO₃⁻, Na⁺, Cl⁻, 3-o-methylglucose, and leucine than did control roots. Stimulatory effects on uptake were more pronounced with freshly cut roots than with roots that were washed and aged. Solutes were accumulated against a concentration gradient, and toxin-treated tissues developed a steeper gradient than did control tissues. Toxin affected both the low and high affinity uptake systems for Na⁺ and Cl⁻. Toxin did not affect uptake of Na₂⁻, K⁺, Ca²⁺, phosphate ion (H₂PO₄⁻ and HPO₄⁻), SO₄⁻, and glutamic acid. No toxin-induced leakage of any solute tested was detected within 5 to 6 hr after initial exposure to toxin. The data suggest that toxin from H. carbonum does not cause the general plasma membrane derangement caused by other host-selective toxins. Instead, H. carbonum toxin may cause specific changes in characteristics of the plasmalemma, which result in increased uptake of certain solutes.